The overall aim of these studies is to further understand the role of POSH (Plenty of SH3s) in apoptotic signaling in neurons and other cells. In response to a number of severe stresses, a signal transduction pathway is engaged leading from Rac to MLK to MKK to JNK and ending in apoptotic cell death. POSH is a recently discovered protein that acts as a molecular scaffold to bind Rac, MLKs and another group of scaffold proteins, JIPs, to facilitate apoptotic signaling through this pathway. This multi-protein signaling complex has been called the POSH-JIP Apoptotic Complex (PJAC). The role of POSH in this pathway appears to be essential; reducing POSH levels has been reported to reduce apoptosis in neurons and neuronally differentiated PC12 cells following nerve growth factor withdrawal, as well as in a rodent model of stroke. Understanding how POSH functions and is regulated is therefore critical to understanding stress-induced apoptosis, and ultimately may allow manipulation of the pathway in clinically useful ways. Over-expression of POSH induces apoptosis in neurons and other cell types examined, but can be prevented by the pro-survival protein kinase, Akt. We have shown that Akt directly phosphorylates POSH and blocks its ability to bind Rac. We have identified major and minor phosphorylation sites in POSH in vitro using mass spectrometry and we propose to use similar techniques to determine Akt phosphorylation sites in POSH in vivo, and to ask how phosphorylation at these sites affects POSH function. We also propose to investigate assembly of the PJAC complex, and ask what components of PJAC are constitutively bound to POSH in viable, unstressed cells, versus cells subjected to apoptotic stress. To further understand PJAC assembly, we will precisely identify the binding sites in POSH for MLK3 and JIP1, using yeast two-hybrid studies and biochemical studies with purified proteins. We also propose to identify critical regions in POSH by mutagenizing POSH and selecting for mutants that no longer induce apoptosis when over-expressed in mammalian cells. Finally, in addition to its function as a protein scaffold, POSH has ubiquitin ligase (E3) activity mediated by a RING domain. We will employ a variety of in vitro ubiquitination assays to test several models of how POSH's E3 activity might regulate apoptotic signaling within the cell. The proposed studies should lead to a greater understanding of how POSH promotes apoptotic signaling in neurons and other cells, and how this process is regulated. These studies are clinically relevant and may ultimately lead to improved therapies for stroke and other degenerative neurological diseases.